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Involvement of TORC2, a CREB co-activator, in the in vivo-specific transcriptional control of HTLV-1.

Jiang S, Inada T, Tanaka M, Furuta RA, Shingu K, Fujisawa J - Retrovirology (2009)

Bottom Line: However, the expression of TORC2, a co-activator of CREB, decreased substantially in the EL4-Gax cells in vivo, and this returned to normal levels in ex vivo culture.The reduced expression of TORC2 was associated with translocation from the nucleus to the cytoplasm.These results suggest that the TORC2 may play an important role in the in vivo -specific transcriptional control of HTLV-1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology Kansai Medical University, Moriguchi, Osaka, Japan. jiangs@takii.kmu.ac.jp

ABSTRACT

Background: Human T-cell leukemia virus type 1 (HTLV-1) causes adult T -cell leukemia (ATL) but the expression of HTLV-1 is strongly suppressed in the peripheral blood of infected people. However, such suppression, which may explain the long latency in the development of ATL, is readily reversible, and viral expression resumes quickly with ex vivo culture of infected T -cells. To investigate the mechanism of in vivo -specific transcriptional suppression, we established a mouse model in which mice were intraperitoneally administered syngeneic EL4 T -lymphoma cells transduced with a recombinant retrovirus expressing a GFP-Tax fusion protein, Gax, under the control of the HTLV-1 enhancer (EL4-Gax).

Results: Gax gene transcription was silenced in vivo but quickly up-regulated in ex vivo culture. Analysis of integrated Gax reporter gene demonstrated that neither CpG methylation of the promoter DNA nor histone modification was associated with the reversible suppression. ChIP-analysis of LTR under suppression revealed reduced promoter binding of TFIIB and Pol-II, but no change in the binding of CREB or CBP/p300 to the viral enhancer sequence. However, the expression of TORC2, a co-activator of CREB, decreased substantially in the EL4-Gax cells in vivo, and this returned to normal levels in ex vivo culture. The reduced expression of TORC2 was associated with translocation from the nucleus to the cytoplasm. A knock-down experiment with siRNA confirmed that TORC2 was the major functional protein of the three TORC-family proteins (TORC1, 2, 3) in EL4-Gax cells.

Conclusion: These results suggest that the TORC2 may play an important role in the in vivo -specific transcriptional control of HTLV-1. This study provides a new model for the reversible mechanism that suppresses HTLV-1 expression in vivo without the DNA methylation or hypoacetylated histones that is observed in the primary cells of most HTLV-1 -infected carriers and a substantial number of ATL cases.

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ChIP analysis of the enhancer/promoter region in Gax provirus. A. Schematic representation of the 5'-LTR in the R3Gaxbsr reporter gene. The three 21 -bp enhancer sequences (boxes), the TATA sequence, and the transcription start site (+1) are shown. Primers for PCR are indicated by arrows. The 5'- and 3'-ends of amplified DNA are denoted as the nucleotide positions relative to the transcription start site. Primers #1 and #2 amplify the enhancer region, and primers #3 and #4 amplify the promoter region of Gax-5'-LTR. B. Binding of CREB, phosphor-CREB and CBP to the Gax enhancer region was constant in EL4-Gax cells in vitro (a) and in vivo (b)(left), but the enhancer binding of Gax was reduced when EL4-Gax cells were grown in vivo (right). C. Expression of CREB protein in EL4-Gax cells. Anti-CREB1, anti-phosphor-CREB antibodies were used to detect proteins in EL4-Gax cells grown in vitro, in vivo, and ex vivo. Equivalent protein loading was confirmed by stripping and re-probing the blot with an anti-β-actin antibody. D. Binding of acetylated histone 3 at Lys-9, 14 (H3), acetylated histone 4 at Lys- 5, 8, 12, 16 (H4) and trimethylated histone 3 at Lys-4 (H3K4tri) to the Gax enhancer region was not changed in EL4-Gax cells either in vitro (a) or in vivo (b), but the promoter binding of the basic transcription factor TFIIB and of RNA polymerase II (Pol-II) was reduced when EL4-Gax cells were grown in vivo. Factors binding to promoters of EF-1a and β-globin are presented as positive and negative controls, respectively.
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Figure 3: ChIP analysis of the enhancer/promoter region in Gax provirus. A. Schematic representation of the 5'-LTR in the R3Gaxbsr reporter gene. The three 21 -bp enhancer sequences (boxes), the TATA sequence, and the transcription start site (+1) are shown. Primers for PCR are indicated by arrows. The 5'- and 3'-ends of amplified DNA are denoted as the nucleotide positions relative to the transcription start site. Primers #1 and #2 amplify the enhancer region, and primers #3 and #4 amplify the promoter region of Gax-5'-LTR. B. Binding of CREB, phosphor-CREB and CBP to the Gax enhancer region was constant in EL4-Gax cells in vitro (a) and in vivo (b)(left), but the enhancer binding of Gax was reduced when EL4-Gax cells were grown in vivo (right). C. Expression of CREB protein in EL4-Gax cells. Anti-CREB1, anti-phosphor-CREB antibodies were used to detect proteins in EL4-Gax cells grown in vitro, in vivo, and ex vivo. Equivalent protein loading was confirmed by stripping and re-probing the blot with an anti-β-actin antibody. D. Binding of acetylated histone 3 at Lys-9, 14 (H3), acetylated histone 4 at Lys- 5, 8, 12, 16 (H4) and trimethylated histone 3 at Lys-4 (H3K4tri) to the Gax enhancer region was not changed in EL4-Gax cells either in vitro (a) or in vivo (b), but the promoter binding of the basic transcription factor TFIIB and of RNA polymerase II (Pol-II) was reduced when EL4-Gax cells were grown in vivo. Factors binding to promoters of EF-1a and β-globin are presented as positive and negative controls, respectively.

Mentions: Enhancer binding of CREB and pCREB was first examined in EL4-Gax cells either in vivo (b) or under in vitro (a) culture conditions. As shown in Figure 3B (lanes 7–10), no significant difference was observed in the amount of CREB or pCREB in complex with the enhancer DNA at the 5'-LTR of the provirus. CBP functions as a cofactor by being tethered to DNA through either pCREB or CREB, in association with Tax, to acetylate histone proteins. Binding of CBP to the HTLV-1 enhancer was observed but showed a similar intensity of protein binding (Fig. 3B, lanes 11, 12).


Involvement of TORC2, a CREB co-activator, in the in vivo-specific transcriptional control of HTLV-1.

Jiang S, Inada T, Tanaka M, Furuta RA, Shingu K, Fujisawa J - Retrovirology (2009)

ChIP analysis of the enhancer/promoter region in Gax provirus. A. Schematic representation of the 5'-LTR in the R3Gaxbsr reporter gene. The three 21 -bp enhancer sequences (boxes), the TATA sequence, and the transcription start site (+1) are shown. Primers for PCR are indicated by arrows. The 5'- and 3'-ends of amplified DNA are denoted as the nucleotide positions relative to the transcription start site. Primers #1 and #2 amplify the enhancer region, and primers #3 and #4 amplify the promoter region of Gax-5'-LTR. B. Binding of CREB, phosphor-CREB and CBP to the Gax enhancer region was constant in EL4-Gax cells in vitro (a) and in vivo (b)(left), but the enhancer binding of Gax was reduced when EL4-Gax cells were grown in vivo (right). C. Expression of CREB protein in EL4-Gax cells. Anti-CREB1, anti-phosphor-CREB antibodies were used to detect proteins in EL4-Gax cells grown in vitro, in vivo, and ex vivo. Equivalent protein loading was confirmed by stripping and re-probing the blot with an anti-β-actin antibody. D. Binding of acetylated histone 3 at Lys-9, 14 (H3), acetylated histone 4 at Lys- 5, 8, 12, 16 (H4) and trimethylated histone 3 at Lys-4 (H3K4tri) to the Gax enhancer region was not changed in EL4-Gax cells either in vitro (a) or in vivo (b), but the promoter binding of the basic transcription factor TFIIB and of RNA polymerase II (Pol-II) was reduced when EL4-Gax cells were grown in vivo. Factors binding to promoters of EF-1a and β-globin are presented as positive and negative controls, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2734550&req=5

Figure 3: ChIP analysis of the enhancer/promoter region in Gax provirus. A. Schematic representation of the 5'-LTR in the R3Gaxbsr reporter gene. The three 21 -bp enhancer sequences (boxes), the TATA sequence, and the transcription start site (+1) are shown. Primers for PCR are indicated by arrows. The 5'- and 3'-ends of amplified DNA are denoted as the nucleotide positions relative to the transcription start site. Primers #1 and #2 amplify the enhancer region, and primers #3 and #4 amplify the promoter region of Gax-5'-LTR. B. Binding of CREB, phosphor-CREB and CBP to the Gax enhancer region was constant in EL4-Gax cells in vitro (a) and in vivo (b)(left), but the enhancer binding of Gax was reduced when EL4-Gax cells were grown in vivo (right). C. Expression of CREB protein in EL4-Gax cells. Anti-CREB1, anti-phosphor-CREB antibodies were used to detect proteins in EL4-Gax cells grown in vitro, in vivo, and ex vivo. Equivalent protein loading was confirmed by stripping and re-probing the blot with an anti-β-actin antibody. D. Binding of acetylated histone 3 at Lys-9, 14 (H3), acetylated histone 4 at Lys- 5, 8, 12, 16 (H4) and trimethylated histone 3 at Lys-4 (H3K4tri) to the Gax enhancer region was not changed in EL4-Gax cells either in vitro (a) or in vivo (b), but the promoter binding of the basic transcription factor TFIIB and of RNA polymerase II (Pol-II) was reduced when EL4-Gax cells were grown in vivo. Factors binding to promoters of EF-1a and β-globin are presented as positive and negative controls, respectively.
Mentions: Enhancer binding of CREB and pCREB was first examined in EL4-Gax cells either in vivo (b) or under in vitro (a) culture conditions. As shown in Figure 3B (lanes 7–10), no significant difference was observed in the amount of CREB or pCREB in complex with the enhancer DNA at the 5'-LTR of the provirus. CBP functions as a cofactor by being tethered to DNA through either pCREB or CREB, in association with Tax, to acetylate histone proteins. Binding of CBP to the HTLV-1 enhancer was observed but showed a similar intensity of protein binding (Fig. 3B, lanes 11, 12).

Bottom Line: However, the expression of TORC2, a co-activator of CREB, decreased substantially in the EL4-Gax cells in vivo, and this returned to normal levels in ex vivo culture.The reduced expression of TORC2 was associated with translocation from the nucleus to the cytoplasm.These results suggest that the TORC2 may play an important role in the in vivo -specific transcriptional control of HTLV-1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology Kansai Medical University, Moriguchi, Osaka, Japan. jiangs@takii.kmu.ac.jp

ABSTRACT

Background: Human T-cell leukemia virus type 1 (HTLV-1) causes adult T -cell leukemia (ATL) but the expression of HTLV-1 is strongly suppressed in the peripheral blood of infected people. However, such suppression, which may explain the long latency in the development of ATL, is readily reversible, and viral expression resumes quickly with ex vivo culture of infected T -cells. To investigate the mechanism of in vivo -specific transcriptional suppression, we established a mouse model in which mice were intraperitoneally administered syngeneic EL4 T -lymphoma cells transduced with a recombinant retrovirus expressing a GFP-Tax fusion protein, Gax, under the control of the HTLV-1 enhancer (EL4-Gax).

Results: Gax gene transcription was silenced in vivo but quickly up-regulated in ex vivo culture. Analysis of integrated Gax reporter gene demonstrated that neither CpG methylation of the promoter DNA nor histone modification was associated with the reversible suppression. ChIP-analysis of LTR under suppression revealed reduced promoter binding of TFIIB and Pol-II, but no change in the binding of CREB or CBP/p300 to the viral enhancer sequence. However, the expression of TORC2, a co-activator of CREB, decreased substantially in the EL4-Gax cells in vivo, and this returned to normal levels in ex vivo culture. The reduced expression of TORC2 was associated with translocation from the nucleus to the cytoplasm. A knock-down experiment with siRNA confirmed that TORC2 was the major functional protein of the three TORC-family proteins (TORC1, 2, 3) in EL4-Gax cells.

Conclusion: These results suggest that the TORC2 may play an important role in the in vivo -specific transcriptional control of HTLV-1. This study provides a new model for the reversible mechanism that suppresses HTLV-1 expression in vivo without the DNA methylation or hypoacetylated histones that is observed in the primary cells of most HTLV-1 -infected carriers and a substantial number of ATL cases.

Show MeSH
Related in: MedlinePlus